We examined the interactive effect of oxidized low density lipoprotein (LDL) and ascorbic acid on collagen production in cultured smooth muscle cells (SMCs). Porcine aortic SMCs were incubated with 50-200 micrograms/ml of human LDL with/without 5 microM Cu2+ for 24 h. Collagen production was assayed by successive salt precipitation at acidic and neutral pH after pepsin digestion of 3H-proline-labeled collagenous protein. Oxidation of LDL was evaluated by electrophoresis and by the level of thiobarbituric acid reactive substances (TBARS). Ascorbic acid reduced the oxidation of LDL + Cu2+ (53% reduction). In the presence of ascorbic acid, no differences were noted in collagen production between LDL and LDL + Cu2+. Without ascorbic acid, collagen production with LDL + Cu2+ was increased dose-dependently up to 6-fold with 150 micrograms/ml LDL, while no such effects were observed at any doses of native LDL. The addition of butylated hydroxytoluene to LDL + Cu2+ strongly suppressed oxidation (88% reduction), and significantly reduced collagen production close to that seen with native LDL. These results indicate that oxidized LDL stimulates collagen production in SMCs, while native LDL does not. Therefore, oxidized LDL may play a direct role in stimulating collagen production in SMCs, which could lead to collagenosis in atherosclerosis.